Enzyme and protein active sites containing two or more interacting metal centers now figure prominently in metallobiochemistry. Systems containing metal clusters composed of Fe atoms (e.g., ferredoxins, sulfite reductase, hemerythrin, ribonucleotide reductase, methane monooxygenase), Cu atoms (e.g., hemocyanin, tyrosinase, ceruloplasmin), Mn atoms (photosystem II, catalase), and more than one element, or heteropolynuclear clusters (e.g., nitrogenase-Mo, Fe; cytochrome oxidase-Fe, Cu; hydrogenase and carbon monoxide dehydrogenase-Ni, Fe), have been identified. The ultimate goals of the proposed research are to elucidate the structures of metal clusters in metalloproteins, to determine the roles these clusters serve in the function of the protein, and to understand how nature designs a cluster for a specific purpose. This knowledge will provide a detailed understanding of biological processes and will aid in the design of pharmaceutical enzyme inhibitors and catalysts for various reactions. This proposal focuses on achieving an understanding of the structure and function of the active site of Ni-containing hydrogenases. Specific goals include: 1. Determining the function of the Ni site and the Ni ligands. 2. Examining the role of Fe, S clusters that are present, and determining the composition of the H2-activating site. 3. Establishing the relationship between the Ni site and active site Fe, S clusters. These goals will be met by using a combination of biophysical techniques and synthetic modelling of the active site. X-ray absorption spectroscopic experiments are proposed in order to address the possible redox role(s) of the Ni site and changes in the ligand environment that occur during catalysis, inhibition, and oxidative deactivation. EPR studies are proposed in order to investigate the relationship of the Ni site with Fe, S clusters. Collaborative Mossbauer experiments are proposed in order to classify the Fe, S clusters present in the enzyme and determine their redox roles. Collaborative ENDOR and IR spectroscopic studies will be involved in investigating the binding of H2 to the Ni site. IR spectroscopy will also be used to investigate the role of thiolate oxidation in the oxidative deactivation of the enzyme. Synthetic model studies are proposed in order to explore the chemistry of Ni thiolate complexes that serve as possible models of the chemistry occurring at Ni or S in the enzyme active site. Substitution of Se in some of these models will provide insight into the role of selenocysteine ligands in hydrogenase. The synthesis of Ni, Fe, S clusters as structural, spectroscopic, and functional models of the active site are also proposed.